Time-resolved fluorometry (TRF) employing long-lifetime emitting luminescent lanthanide chelates has been applied in many specific binding assays, such as e.g. immunoassays, DNA hybridization assays, receptor-binding assays, enzymatic assays, bio-imaging such as immunocytochemical, immunohistochemical assays or cell based assays to measure wanted analyte at very low concentration. Moreover, lanthanide chelates have been used in magnetic resonance imaging (MRI) and position emission tomography (PET)
For TRF application, an optimal label has to fulfill several requirements. First, it has to be photochemically stable both in the ground state and in the excited state and it has to be kinetically and chemically stable. The excitation wavelength has to be as high as possible, preferable over 300 nm. It has to have efficient cation emission i.e. high luminescence yield (excitation coefficient x quantum yield, ϵΦ). The observed luminescence decay time has to be long, and the chelate has to have good water solubility. For the purpose of labeling, it should have a reactive group to allow covalent attachment to a biospecific binding reactant, and the affinity and nonspecific binding properties of the labeled biomolecules have to be retained.
The challenge is to prepare a chelate label to fulfill all requirements in one molecule, and therefore, certain compromises are generally made in the development of suitable labels. As a consequence hereof, a number of attempts (see e.g. the review in Bioconjugate Chem., 20 (2009) 404) have been made to tune the photo-physical properties of the chelate labels suitable for time-resolved fluorometric applications.
One generally used method to improve luminescence intensity is to prepare chelate ligands with several independent chromophoric moieties combined in structure designs, which offer high stabilities and luminescence quantum yields. Chelates which contain two and three separate 4-(phenylethynyl)pyridines are published by Takalo, H., et al., 1996, Helv. Chim. Acta., 79, 789. More recent examples of lanthanide chelates and chelating ligands are those disclosed in e.g. EP 1 447 666, WO 2010/055207, WO 2010/006605 and WO 2008/020113. Based on chelate stability studies with cyclic azamacrocycles (such as DOTA), higher stabilities over open chain chelates (such as DPTA) has been observed, and thus, the main focus with disclosed chelates with three chromophores has been on azamacrocycles tethered to the various chromophores.
Lately, high lanthanide chelate stabilities have been observed with open chain ligands utilizing several 1-hydroxy-2-pyridinone and salisylamide groups although these disclosed chelates are normally eight dentate and do not contain carboxylic acids for coordination to a lanthanide ion, high luminescence and stabilities have been obtained. However, those lanthanide chelates have only moderate total molar absorptivity i.e. below 27,000 with four chromophores, whereas e.g. chelates with only one phenylethynylpyridine subunit normally have absorptivities of 25,000-35,000 cm-1 (Latva, M, et al., 1997, J. Luminescence, 75, 149) depending on the substituents in the chromophore.
A well-known challenge with chelates and ligands having many chromophores is to find out a suitable structure design, which offers high water solubility and at the same time being inert towards any possible bioprocesses. It is known, that the addition of chromophores decreases the solubility of ligands and chelates in water, increases the formation of biospecific binding reactant aggregates during the labeling process and non-specific binding properties of labeled biomolecules. Aggregates will produce purification problems and reduced yield of labeled material. Moreover, increased non-specific binding of labeled biomolecule will enhance back-ground luminescence of biospecific assays and thus reduces assay sensitivity.